RNAI as a Tool to Downregulate Gene Expression
Gene targeting by homologous recombination is commonly used to determine gene function in mammals, but this is a costly and time-consuming process. Alternatively, the functions of many genes can be determined after mRNA inhibition with ribozyme or antisense technologies, Although successful in some situations these technologies have been difficult to apply universally. The advent of siRNA-directed “knockdown” has sparked a revolution In somatic cell genetics, allowing the inexpensive and rapid analysis of gene function in mammals.
Establishing a convenient and reliable method to knock-out gene expression at the mRNA level has been a recurrent theme in molecular biology over the last 15 years. In efforts to generate loss-of function cells or organisms, various molecules that included, for example, antisense sequences, ribozymes, and chimeric oligonucleotides have been tested, but the design of such molecules was based on trial and error, depending on the properties of the target gene.
Moreover, the desired effects were difficult to predict, and often only weak suppression achieved (Braasch & Corey, 2002).
After the discovery of the phenomenon in plants in the early 1990s, in 1998 Andy Fire and Craig Mello for the first time demonstrated with the worm Caenorhabditis elegans that dsRNA (double-stranded RNA) may specifically and selectively inhibit gene expression in an extremely efficient manner (Fire et al., 1998). In their experiment, the sequence of the first strand (the so-called sense RNA) coincides with that of the corresponding region of the target messenger RNA (mRNA). The second strand (antisense RNA) is complementary to this mRNA. The resulting dsRNA turned out to be far more (several orders of magnitude) efficient than the corresponding single-stranded RNA molecules (in particular, antisense RNA). Fire et al., 1998 named the phenomenon RNAi for RNA interference. This powerful gene silencing mechanism has been shown to operate in several species among most phylogenetic phyla.
RNAi begins when an enzyme named DICER encounters dsRNA and chops it into pieces called small-interfering RNAs or siRNAs. This protein belongs to the RNase III nuclease family. A complex of proteins gathers up these RNA remains and uses their code as a guide to search out and destroy any RNAs in the cell with a matching sequence, such as target mRNA (for review see Bosher & Labouesse, 2000).
The RNAi phenomenon (Akashi et al., 2001) might be summarized as follows:                Step 1: dsRNA recognition and scanning process.        Step 2: dsRNA cleavage through RNase III activity and production of siRNAs.        Step 3: association of the siRNAs and associated factors in RISC complexes.        Step 4: recognition of the complementary target mRNA.        Step 5: cleavage of the target mRNA in the centre of the region complementary to the siRNA.        Step 6: degradation of the target mRNA and recycling of the RISC complex.        
In trying to apply the RNAI phenomenon as a technology for gene knockdown, it was soon realized that mammalian cells have developed various protective phenomena against viral infections that could impede the use of this approach. Indeed, the presence of extremely low levels of viral dsRNA triggers an interferon response, resulting in a global non-specific suppression of translation, which in turn triggers apoptosis (Williams, 1997, Gil & Esteban, 2000).
In 2000, a first attempt with dsRNA resulted in the specific inhibition of 3 genes (MmGFP under the control of the Elongation Factor 1a, E-cadherin, and c-mos) in the mouse oocyte and early embryo. Translational arrest, and thus a PKR response, was not observed as the embryos continued to develop (Wianny & Zernicka-Goetz, 2000). One year later, research at Ribopharma AG (Kulmbach, Germany) first demonstrated the functionality of RNAi in mammalian cells. Using short (20-24 base pairs) dsRNAs—which are called SIRPLEX™—they specifically switched off genes even in human cells without initiating the acute-phase response. Similar experiments carried out later by other research groups (Elbashir et al., 2001; Caplen et al., 2001) further confirmed these results.
A year later, Paddison et. al. (Paddison et al, 2002) tried to use small RNAs folded in hairpin structures to inhibit the function of specific genes. This work was inspired by previous studies showing that some genes in Caenorhabditis elegans naturally regulate other genes through RNAi by coding for hairpin-structured RNAs. Tested in a variety of normal and cancer human and mouse cell lines, short hairpin RNAs (shRNAs) are able to silence genes as efficiently as their siRNA counterparts. Moreover, shRNAs exhibit better reassociation kinetics in vivo than equivalent duplexes. Even more important, these authors generated transgenic cell lines engineered to synthesize shRNAs that exhibit a long-lasting suppressing effect throughout cell divisions (Eurogentec). Recently, another group of small RNAs (also comprised in the range of 21-25 nt) was shown to mediate downregulation of gene expression. These RNAs, known as small temporally regulated RNAs (stRNAs), have been described in Caenorhabditis elegans were they regulate timing of gene expression during development. It should be noted that stRNAs and siRNAs, despite obvious similarities, proceed through different modes of action (for review see Banedjee & Slack, 2002. In contrast with siRNAs, 22 nt long stRNAs downregulate expression of target mRNA after translational initiation without affecting mRNA integrity. Recent studies indicate that the two stRNAs first described in nematodes are the members of a huge family with hundreds of additional micro-RNAs (miRNAs) existing in metazoans (Grosshans & Slack, 2002).
Scientists have initially used RNAi in several systems, including Caenorhabditis elegans, Drosophilia, trypanosomes, and various other invertebrates. Moreover, using this approach, several groups have recently presented the specific suppression of protein biosynthesis in different mammalian cell lines—specifically in HeLa cells—showing that RNAi is a broadly applicable method for gene silencing in vitro. Based on these results, RNAi has rapidly become a well recognized tool for validating (identifying and assigning) gene functions. RNA interference employing short dsRNA oligonucleotides will, moreover, permit to decipher the function of genes being only partially sequenced. RNAi will therefore become inevitable in studies such as:                Inhibition of gene expression at the post-transcriptional level in eukaryotic cells. In this context, RNAi is a straight-forward tool to rapidly assess gene function and reveal null phenotypes.        Development of the RNAi technology for use in post-implantation embryos.        The predominant economic significance of RNA interference is established by its application as a therapeutic principle. As so, RNAi may yield RNA-based drugs to treat human diseases.        
Glaucoma
Glaucoma is one of the leading causes of blindness, Approximately 15% of cases of blindness world-wide result from glaucoma. The most common type, primary open-angle glaucoma, has a prevalence of 1/200 in the general population over 40 years of age.
Glaucoma has been simply defined as the process of ocular tissue destruction caused by a sustained elevation of the Intra Ocular Pressure (IOP) above its normal physiological limits.
It is becoming increasingly clear that many forms of glaucoma have a genetic component, and much current research is focused on identifying chromosomal regions and genes that contribute to glaucoma. It is likely that the aetiology of OAG is multifactorial, resulting from a combination of mutations in more than one gene and as yet unidentified environmental factors. With regard to juvenile and adult-onset OAG, several loci have been identified. However, only one gene is known, namely the myocilin/TIGR (trabecular meshwork inducible glucocorticoid response) gene at the GLC1A locus on chromosome 1q21-q31. More than thirty mutations of this gene have been identified in ethnically diverse populations worldwide. Studies have shown that it is responsible for only about 5% of OAG overall (See reviews in Wirtz & Samples, 2003, and Khaw et al, 2004a).
Pathogenesis
Most glaucomas are characterised by an elevated IOP, although the level of elevation may vary. In those glaucomas in which the elevation is initially low (i.e., open angle glaucoma, melanocytic glaucoma) and some secondary glaucoma, retinal ganglion cell and optic nerve damage are slow to progress. In angle-closure glaucoma the sudden high rise in IOP often renders the eye blind, undoubtedly primarily due to a cessation of axoplasmic flow at the level of the lamina cribrosa.
In human studies, it has been widely accepted that tissue ischaemia has a part to play in the initiation or progression of the optic disc damage that occurs in glaucoma. Retinal ganglion cell degeneration may be necrosis, but the possibility that it is apoptosis triggered by the rise in IOP is plausible, and the respective roles of nitric oxide and glutamate are thought to be relevant during progression of the disease (For a recent review on the subject see Osborne et al, 2003).
Treatment
Although several aetiologies are involved in the glaucoma complex, the absolute determinant in therapy selection is the amount of primary and/or induced change in pressure within the iridocorneal angle.
Current therapies include medications or surgeries aimed at lowering this pressure, although the pathophysiological mechanisms by which elevated IOP leads to neuronal damage in glaucoma are unknown.
Medical suppression of an elevated IOP can be attempted using four types of drugs: the aqueous formation suppressors (among them, carbonic anhydrase inhibitors, beta-adrenergic blocking agents, or alpha2-adrenoreceptor agonists) miotics (i.e. parasympathomimetics—cholinergics-, or anticholinesterase inhibitors); uveoscleral outflow enhancers; and the hyperosmotic agents (that produce an osmotic pressure gradient across the blood/aqueous barrier within the ciliary epithelium). All four are used in the treatment of glaucoma, the first three commonly as emergency treatment and in long term control while the hyperosmotic agents are invaluable as emergency and preoperative treatment. A fifth category of drugs, the neuroprotection agents, is beginning to emerge as an important possible addition to medical therapy. Indeed, observation that the NOS and glutamate levels are elevated in glaucoma and that they are involved in retinal ganglion cell necrosis or apoptosis has raised the possibility of neuroprotective therapies and even neuroregeneration. Thus NOS inhibitors, exciting amino acid antagonists, glutamate receptor antagonists, apoptosis inhibitors and calcium channel blockers are all potential candidates in the development of future glaucoma therapies. The calcium channel blockers may reduce the effect of impaired microcirculation to the optic nerve head whilst potentially increasing outflow facility at the level of the trabecular cells.
Reviews of various eye disorders and their treatments are given in the references, in particular in Bunce (2005), Costagliola (1995, 2000), Cullinane (2002), Sakaguchi (2002), Shah (2000), and Wang (2005).
Currently our existing therapies must fall short of the mark and the practical difficulties associated with the assessment of outflow facility, the accurate monitoring of therapy and the complexity of surgical techniques all combine to confound the prognosis. The overriding factor in all glaucoma is the degeneration of the retinal ganglion cell, thus neuroprotection through effective ocular hypotension is the essential requirement of any therapy we utilise (for a recent review on the subject, see Khaw et al 2004b).